Modified j-chain

ABSTRACT

The present invention concerns modified recombinant J-chain polypeptides, binding molecules, such as antibodies, comprising the same, and their uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/301,366, filed Sep. 30, 2016, which is a U.S. National Stage entry ofPCT Patent Application No. PCT/US2015/024149, filed on Apr. 2, 2015, andalso claims priority benefit of the filing date of U.S. ProvisionalApplication No. 61/974,738, filed Apr. 3, 2014, the disclosures of whichapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention concerns modified recombinant J-chain polypeptidesand binding molecules, such as antibodies comprising the same.

BACKGROUND OF THE INVENTION

J-chain is an acidic 15-kDa polypeptide, which is associated withpentameric IgM and dimeric IgA via disulfide bonds involving thepenultimate cysteine residue in the 18-amino acid secretory tail-piece(tp) at the C-terminus of the IgM μ or IgA α heavy chain. The threedisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, andCys 108 and 133, respectively. See, e.g. Frutiger et al. 1992,Biochemistry 31, 12643-12647. Structural requirements for incorporationof the J-chain into human IgM and IgA and for polymeric immunoglobulinassembly and association with the J-chain are reported by Sorensen etal. 2000, Int. Immunol. 12(1): 19-27 and Yoo et al. 1999, J. Biol. Chem.274(47):33771-33777, respectively. Recombinant production of solubleJ-chain in E coli is reported by Redwan et al. 2006, Human Antibodies15:95-102.

Methods for making hybrid IgA/IgG and IgM/IgG antibodies are known inthe art. Thus, recombinant production of hybrid IgA2/IgG1 antibodies isreported in Chintalacharuvu et al. 2001, Clin Immunol 101(1):21-31. Ithas been reported that addition of αtp or μtp at the end of IgG γ heavychain facilitates polymerization and enhances effector function such ascomplement activation (Smith et al., J Immunol 1995, 154:2226-2236). TheIgA/IgG hybrid antibodies possess properties of both IgA and IgG.

Despite the advances made in the design of antibodies, there remains aneed for modified antibodies with improved properties, such as improvedaffinity, specificity and/or avidity.

As the field has progressed, antibody function has been enhanced throughcreative means of protein engineering, such as to provide higheraffinity, longer half-life, and/or better tissue distribution, as wellas combination of small and large molecule technologies for increasedfocus of cell destruction via toxic payload delivery (e.g. antibody-drugconjugates). Another approach to improving antibody function takesadvantage of the bivalent binding of the immunoglobulin G (IgG)structure which allows one IgG molecule to bind two antigens. Indeed, incertain applications, there exists good potential for asymmetricantibodies to exert useful functions by simultaneously binding twodifferent target antigens. To address this need, a variety of constructshave been produced to yield a single molecule that can bind twodifferent antigens, allowing for functions never before seen in nature.An example of this bi-specific approach is “blinatumomab” (MT103 orAMG103) which binds the CD3 and CD19 receptors, on T- and B-cells,respectively. This tethering of a cytotoxic T-cell to a cancerousB-cell, allows for effective treatment of B-cell leukemia.

However, there remain significant technical difficulties inconstruction, expression and production of bispecific antibodies.Although bispecific antibodies are regarded as promising therapeuticagents due to their ability to simultaneously bind two differentantigens, their utility is limited due to problems with stability andmanufacturing complexity.

Various forms of protein engineering have been used to matchheterologous heavy chains, plus appropriate pair-wise matching of heavyand light chains to efficiently yield a bi-specific IgG. In addition,various bispecific antibody formats, including quadromas, chemicalheteroconjugates, recombinant constructs using selectedheterodimerization domains and recombinant constructs of minimal sizeconsisting of two minimal antigen-binding sites.

However, all of these efforts have been fraught with difficulty.

Thus, despite efforts directed toward the development of engineered,such as bispecific, antibodies, there remains a great need fordeveloping more efficient platforms. This is particularly true in thecase of therapeutic antibodies, where the design and production of new,modified, antibodies and antibody-like molecules with multiplespecificities can shorten the timeline between discovery and clinicalintroduction of such therapeutics.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the recognitionthat the J-chain of an IgM or IgA antibody can be modified byintroducing one or more binding moieties into a native J-chain sequence,and the modified J-chain can be introduced into IgM, IgA, IgG/IgM orIgG/IgA antibodies without compromising the functionality of therecipient antibody or the binding of the modified J-chain to its target.This allows the modified J-chain with binding moiety to interact withone set of target antigens, while the IgA, IgM, IgG/IgM or IgG/IgAantibody can react with a different set of target antigens.

The invention is further based on the recognition that by directing themodified J-chain to an effector cell, such as a T-cell, NK-cell,macrophage or neutrophil, the immune response of the body can beactivated, and the antibody dependent cellular cytotoxicity (ADCC)response can be improved.

In one aspect, the present invention concerns a modified J-chaincomprising an extraneous binding moiety introduced into a nativesequence J-chain.

In one embodiment, the native sequence J-chain is the native humanJ-chain sequence of SEQ ID NO: 1, or a functional fragment thereof.

In another embodiment, the extraneous binding moiety is introduced intothe native human J-chain sequence of SEQ ID NO: 1 by direct or indirectfusion.

In yet another embodiment, binding moiety is introduced by indirectfusion through a peptide linker.

In one embodiment, the indirect fusion is accomplished through a peptidelinker at or around the C- and/or N-terminus of the binding moiety.

In another embodiment, the extraneous binding moiety is introduced intothe native human J-chain sequence of SEQ ID NO: 1 at or around theC-terminus and/or the N-terminus, such as within about 10 residues fromthe C-terminus and/or the N-terminus.

In a further embodiment, the extraneous binding moiety is introducedinto the native human J-chain sequence in between cysteine residues 92and 101 of SEQ ID NO: 1.

In a still further embodiment, the extraneous binding moiety isintroduced into the native human J-chain sequence of SEQ ID NO: 1 at ornear a glycosylation site.

The peptide linker, if present, may, for example, be about 10 to 20amino acids long, or about 15 to 20 amino acids long, or 15 amino acidslong.

In a further embodiment, the extraneous binding moiety is introducedinto the native human J-chain sequence of SEQ ID NO: 1 by chemical orchemo-enzymatic derivatization. The chemical linker may be a cleavableor a non-cleavable linker, where the cleavable linker may, for example,be a chemically labile linker or an enzyme-labile linker.

In a further embodiment, the linker is selected from the groupconsisting of N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT),bifunctional derivatives of imidoesters, active esters, aldehydes,bis-azido compounds, bis-diazonium derivatives, diisocyanates, andbis-active fluorine compounds.

In a different embodiment, the J-chain is modified by insertion of anenzyme recognition site, and by post-translationally attaching anextraneous binding moiety at the enzyme recognition site through apeptide or non-peptide linker.

In all embodiments, the extraneous binding moiety may, for example, beselected from the group consisting of antibodies, antigen-bindingfragments of antibodies, antibody-drug conjugates, antibody-likemolecules, antigen-binding fragments of antibody-like molecules, solubleand membrane-bound proteins, ligands, receptors, virus-like particles,protein toxins, enzymes, and alternative scaffolds. Examples ofalternative scaffolds include darpins, fibronectin domains, adnectins,and knottins. Typical antigen-binding fragments include F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv, and single domain antibodies. In a preferredembodiment, the antigen-binding fragment is scFv.

In one embodiment, the extraneous binding moiety of the modified J-chainbinds to an effector cell, where the effector cell may, for example, beselected from the group consisting of T-cells, natural killer (NK)cells, macrophages and neutrophils.

In one embodiment, the effector cell is a T-cell, where the extraneousbinding moiety may, for example, bind to CD3ε on the T-cell.

In another embodiment, the effector cell is an NK cell, where the targetfor the extraneous binding moiety of the modified J-chain may, forexample be selected from the group consisting of CD16, CD64 and NKG2D onthe NK cell.

In a yet another embodiment, the effector cell is a macrophage, wherethe extraneous binding moiety of the modified J-chain may, for example,bind to CD14 on the macrophage.

In a further embodiment, the effector cell is a neutrophil, where theextraneous binding moiety of the modified J-chain may, for example, bindto CD16b or CD177 on the neutrophil.

In another aspect, the invention concerns an antibody comprising amodified J-chain as hereinbefore described, or an antigen-bindingfragment of such antibody. The antibody can be an IgM, IgA, IgG/IgM orIgG/IgA antibody, and includes multi-specific, e.g. bispecificantibodies.

In one embodiment, the antibody is an IgM antibody, an IgA antibody, oran IgG antibody comprising a tail piece, or an antigen-binding fragmentthereof.

In another embodiment, the antibody has binding specificity to one ormore binding target selected from the group consisting of target cells,soluble binding targets, cell surface receptors, matrix proteins,transporter receptors.

In yet another embodiment, the antibody binds to a tumor cell.

In a further embodiment, the antibody binds to a tumor target associatedantigen listed in FIG. 19, where the J-chain may, for example, bemodified to bind any of the effector cell targets listed in FIG. 19.

In a still further embodiments, the modified J-chain is present in theantibodies in a V-linker-J orientation or in a J-linker-V orientation.

In one embodiment, the tumor is a hematologic cancer or a solid tumor,where the hematologic cancer may, for example, be leukemia, lymphoma,myeloma, and myelodisplastic syndrome, specifically including acutemyeloid leukemia, acute lymphoblastic leukemia, chronic myelogenousleukemia, or chronic lymphocitic leukemia, Hodgkin's lymphoma andnon-Hodgkin's lymphoma. In such embodiments, the antibody may, forexample, bind to one or more of CDIM, CD19, CD20, CD22, CD33, CD70,CD56, CD138, and the modified J-chain may bind to CD3ε.

In another embodiment, the tumor is an epithelial tumor.

In yet another embodiment, the antibody binds to a carbohydrate-basedtarget on the tumor.

In a further embodiment, the antibody binds to a viral antigen, such asan HBV antigen or an HIV antigen, e.g. PreS1 or GP120.

In a further aspect, the invention concerns a composition comprising theIgM, IgA, IgG/IgM, IgG/IgA antibodies comprising a modified J-chain, asdescribed. The composition may, for example, be a pharmaceuticalcomposition or a diagnostic composition.

In a still further aspect, the invention concerns a method of treating atumor or viral disease comprising administering to a subject in need aneffective amount of an IgM, IgA, IgG/IgM, IgG/IgA antibody with amodified J-chain, as described herein.

In another aspect, the invention concerns the use of an IgM, IgA,IgG/IgM, IgG/IgA antibody with a modified J-chain, as described hereinin the preparation of a medicament for the treatment of a tumor or viraldisease.

In yet another aspect, the invention concerns the use of an IgM, IgA,IgG/IgM, IgG/IgA antibody with a modified J-chain, as described hereinin the treatment of a tumor or viral disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of an IgM pentamer, comprising aJ-chain, wherein chains A and B are identical in native IgM.

FIG. 2 shows the schematic structures of IgA, dimeric IgA, and secretoryIgA (sIgA).

FIG. 3 shows the amino acid sequence of mature human J-chain (SEQ ID NO:1).

FIG. 4 shows the alignment of mature human J-chain (SEQ ID NO: 1) andJ-chains from various animal species (SEQ ID Nos: 1 to 44).

-   Hum=Human NP_653247 23-159 (SEQ ID NO: 1)-   Chm=Chimpanzee XP_001160135 39-175 (SEQ ID NO: 2)-   Gor=Gorilla XP_004038830 23-159 (SEQ ID NO: 3)-   Ora=Orangutan NP_001125381 23-159 (SEQ ID NO: 4-   Gib=Gibbon XP_003265761 39-175 (SEQ ID NO: 5)-   Mar=Marmoset XP_003732538 32-168 (SEQ ID NO: 6)-   Cyn=Cynomolgus Monkey NP_001247815 23-159 (SEQ ID NO: 7)-   Bab=Baboon XP_003898844 23-159 (SEQ ID NO: 8)-   Squ=Squirrel Monkey XP_003931978 23-159 (SEQ ID NO: 9)-   Shr=Tree Shrew XP_006142955 25-160 (SEQ ID NO: 10)-   Dol=Dolphin XP_004328961 25-158 (SEQ ID NO: 11)-   Kil=Killer Whale XP_004283629 25-158 (SEQ ID NO: 12)-   Ele=Elephant XP_003414110 25-158 (SEQ ID NO: 13)-   Sea=Seal XP_006729388 25-158 (SEQ ID NO: 14)-   Rhi=Rhinoceros XP_004419202 25-157 (SEQ ID NO: 15)-   Cat=House Cat XP_003985350 25-158 (SEQ ID NO: 16)-   Wol=Wolf XP_532398 26-159 (SEQ ID NO: 17)-   Pan=Giant Panda EFB23253 1-134 (SEQ ID NO: 18)-   Fer=Ferret XP_004766376 26-158 (SEQ ID NO: 19)-   Hor=Horse NP_001271464 25-158 (SEQ ID NO: 20)-   Gui=Guinea Pig NP_001265705 25-160 (SEQ ID NO: 21)-   Cam=Camel XP_006188738 25-158 (SEQ ID NO: 22)-   Goa=Goat XP_005681786 25-158 (SEQ ID NO: 23)-   Chn=Chinchilla XP_005392838 94-229 (SEQ ID NO: 24)-   Ham=Hamster XP_005068228 24-160 (SEQ ID NO: 25)-   She=Sheep XP_004009937 25-158 (SEQ ID NO: 26)-   BBa=Brown Bat XP_006094475 25-158 (SEQ ID NO: 27)-   Ant=Antelope XP_005983836 25-158(SEQ ID NO: 28)-   Cow=Cow NP_786967 25-157 (SEQ ID NO: 29)-   Mou=Mouse NP_690052 23-159 (SEQ ID NO: 30)-   Rat=Rat EDL88516 23-159 (SEQ ID NO: 31)-   Hed=Hedgehog XP_004703237 25-157 (SEQ ID NO: 32)-   Rab=Rabbit P23108 1-136 (SEQ ID NO: 33)-   Opo=Opossum XP_003341415 29-162 (SEQ ID NO: 34)-   All=Alligator XP_006270094 26-159 (SEQ ID NO: 35)-   Tur=Turtle XP_005304167 26-159 (SEQ ID NO: 36)-   Tas=Tasmania Devil XP_003772863 27-160 (SEQ ID NO: 37)-   Pla=Platypus XP_003430954 36-160 (SEQ ID NO: 38)-   Par=Parakeet XP_005142787 28-160 (SEQ ID NO: 39)-   Duc=Duck XP_005031370 28-160 (SEQ ID NO: 40)-   Chi=Chicken NP_989594 26-158 (SEQ ID NO: 41)-   Tur=Turkey XP_003205717 27-159 (SEQ ID NO: 42)-   Fal=Falcon XP_005243236 29-160 (SEQ ID NO: 43)-   Spa=Sparrow XP_005492217 29-158 (SEQ ID NO: 44)

FIG. 5 shows the alignment of the amino acid sequence of human and bat(back flying Pteropus alecto) J-chain amino acid sequences (SEQ ID NOs:1 and 45, respectively).

FIG. 6: An IgM antibody lacking its J-chain (negative control) and IgMcomprising a J-chain were separated on reducing SDS PAGE and detected byWestern Blot using an anti-J-chain antibody. The anti-J-chain antibodyonly reacted with the IgM comprising a J-chain.

FIG. 7: Harvested supernatant from transfected CHO cells were dividedequally for immunoprecipitation with either anti-mu or anti-myc affinitymatrix. These CHO cells were expressing an IgM with anti-B cellsspecificity known as CDIM (for cell death inducing molecule). This IgMantibody is known as IGM-55.5. The immunoprecipitated proteins wereseparated on reducing SDS PAGE and detected by Western Blot by anti-J oranti-myc antibodies. Both N-terminal tagged anti-CD3scFvJ and C-terminaltagged J-antiCD3scFv transfected cells displayed a positive band around51 kD, which reacted to both anti-J and anti-myc antibodies.

FIG. 8 illustrates two different orientations of a bis-specific IgMpentamer comprising a modified J-chain with binding specificity for aT-cell (the T-cell antigen CD3).

FIG. 9: Sequence (SEQ ID NO: 46) and structure of an anti-CD3single-chain Fv comprising a J-chain at the C-terminus (antiCD3scFv-J).SP=signal peptide.

FIG. 10: Sequence (SEQ ID NO: 47) and structure an anti-CD3 single-chainFv comprising a J-chain at the N-terminus (J-antiCD3scFv). SP=signalpeptide.

FIG. 11 is a schematic illustration of an asymmetric bi-specific IgMpentamer with binding-specificity for a target antigen, comprising abinding domain covalently attached to the J-chain with bindingspecificity for an effector cell.

FIG. 12 shows Western blots of an anti-CD20 IgM pentamer with a J-chaincovalently modified with an CD3 scFv binding domain. Non-reducing PAGEstained with Coomassie Blue observes the full size of IgM: approximately1 million daltons MW. The modified J-chain incorporates into the IgMpentamers in both orientations: CD3-J and J-CD3.

FIG. 13 shows the results of transient co-transfections of the heavychain (HC), light chain (LC), and modified J-chain of a bispecificanti-CD20 IgM (CD20_(IgM)) with a modified J-chain providing bindingspecificity to CD3 (CD3_(J)).

FIG. 14 shows that incorporation of a modified J-chain into an anti-CD20IgM has no negative effect on CDC activity. In addition, the data showthat an IgM molecule comprising a modified J-chain in either orientation(V15J or J15V, where “15” designates the length of the linker) issignificantly more potent than an anti-CD20 IgG antibody.

FIG. 15 shows that a bispecific CD20_(IgM)×CD3_(J-chain) molecule ishighly potent at B-cell depletion as a result of active T-cell killingof B-cells due to the CD3-specificity of the modified J-chain. Raji, aCD19+CD20+ B cell line, was co-cultured with T-ALL, a CD8+ Cytolytic Tcell line) with CD20 IgM×J-wild-type or CD20 IgM×CD3-J chain for 24hours at 37 degrees, 5% CO2. Cell were harvested and stained withfluorescent antibodies to CD3 and CD19 and analyzed by flow cytometry toassess viable B cells.

FIG. 16 shows that a bispecific anti-CD20 IgM molecule with a modifiedJ-chain providing binding specificity to CD3 has significantly improvedcytotoxic activity over an anti-CD20 IgM pentamer with wild-typeJ-chain. The T-cell engaging bispecific IgM pentamer is effective at aconcentration of 1 ng/ml, and is 300-times more potent compared toanti-CD20 IgM with wild-type J-chain.

FIG. 17 shows in vivo B-cell depletion with anti-CD20 IgG, IgM andbispecific IgM. Humanized NSG mice reconstituted with human CD34+ stemcells (Jackson Laboratory; Stock Number: 007799) were purchased andwhole blood was analyzed for pre-dose levels of CD19+ B cells. CD20IgM×CD3-J chain was prepared for intravenous dosing animals at 0.5mg/kg. Following 6 hours after dosing, whole blood was obtained fromanimals and analyzed by flow cytometry for circulating levels of human Bcells using a fluorescently labeled CD19+ antibody.

FIG. 18 is a Western blot of a CD16 specific Vhh camelid-J-chain.

FIG. 19 lists IgM antibody targets and effector cell targets for amodified J-chain. Any of the IgM antibody targets listed in the leftcolumn of the table can be combined with any of the modified J-chaintargets listed in the right column.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges encompassed within the invention, subject to anyspecifically excluded limit in the stated range.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provides one skilled in the art with a general guide to manyof the terms used in the present application.

All publications mentioned herein are expressly incorporated herein byreference to disclose and describe the methods and/or materials inconnection with which the publications are cited.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv).The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The basic 4-chain antibody unit is a heterotetramericglycoprotein composed of two identical light (L) chains and twoidentical heavy (H) chains. Unless noted otherwise, the term “antibody”is used herein in the broadest sense and specifically includes allisotypes, sub-classes and forms of antibodies, including IgG, IgM, IgA,IgD, and IgE antibodies and their fragments, preferably antigen-bindingfragments. Preferred antibodies herein include IgM and IgA antibodiesand their antigen-binding fragments, which may be modified to includesequences from other isotypes, such as IgG to produce chimericantibodies.

In the case of IgGs, the 4-chain unit is generally about 150,000daltons. Each L chain is linked to an H chain by one covalent disulfidebond, while the two H chains are linked to each other by one or moredisulfide bonds depending on the H chain isotype. Each H and L chainalso has regularly spaced intrachain disulfide bridges. Each H chain hasat the N-terminus, a variable domain (V_(H)) followed by three constantdomains (C_(H)) for each of the α and γ chains and four C_(H) domainsfor μ and ε isotypes. Each L chain has at the N-terminus, a variabledomain (V_(L)) followed by a constant domain at its other end. The V_(L)is aligned with the V_(H) and the C_(L) is aligned with the firstconstant domain of the heavy chain (C_(H1)). Particular amino acidresidues are believed to form an interface between the light chain andheavy chain variable domains. The pairing of a V_(H) and V_(L) togetherforms a single antigen-binding site.

IgM is a glycoprotein which forms polymers where multipleimmunoglobulins are covalently linked together with disufide bonds. IgMmostly exists as a pentamer but also as a hexamer and therefore contains10 or 12 antigen binding sites. The pentameric form typically containsan additional polypeptide, called the J-chain, but can also be made inthe absence of J-chain. The pentameric IgM molecule has a molecularweight of approximately 970 kDa. Due to its polymeric nature, IgMpossesses high avidity and is particularly effective in complementactivation. Unlike in IgG, the heavy chain in IgM monomers is composedof one variable and four constant domains. The IgM constant domains aredesignated herein as CM1 or Cμ1, CM2 or Cμ2, CM3 or Cμ3, and CM4 or Cμ4,wherein the “CM” and Cμ” designations are used interchangeably. Thestructure of an IgM pentamer is illustrated in FIG. 1.

The term “IgM” is used herein in the broadest sense and specificallyincludes mono-, and multi-specific (including bispecific) IgM molecules,such as, for example, the multi-specific IgM binding molecules disclosedin PCT Application No. PCT/US2014/054079, the entire disclosure of whichis expressly incorporated by reference herein.

The term “IgM binding unit” or “IgM antibody binding unit” is used inthe broadest sense and specifically covers an IgM antibody heavy chainconstant region polypeptide, comprising at least a CM4 constant domain,fused to a variable domain sequence (V_(H)) binding to a target (e.g.antigen), with or without an associated antibody light chain variabledomain (V_(L)) sequence.

The term “bispecific IgM binding unit” or “bispecific IgM antibodybinding unit” is used in the broadest sense and specifically covers apair of IgM antibody heavy chain constant region polypeptides,comprising at least a CM4 constant domain, fused to a variable domainsequence (V_(H)), each variable domain sequence binding to a differenttarget, with or without associated antibody light chain variable domain(V_(L)) sequences. In one embodiment, the bispecific IgM antibodycomprises two V_(H)V_(L) antigen binding regions, each capable ofbinding to a different epitope on one antigen or epitopes on twodifferent antigens. The bispecific IgM antibody binding units can befull length from a single species, or be chimerized or humanized. Thebispecific IgM antibodies of the present invention have a penta- orhexameric ring structure comprising five or six bispecific IgM bindingunits.

The term “multi-specific IgM” is used herein in the broadest sense torefer to IgM antibodies with two or more binding specificities. Thus,the term “multi-specific” includes “bispecific”, e.g. bispecificantibodies or bispecific binding units, including IgM pentamerscomprising at least two monospecific subunits, each binding to adifferent antigen (AA, BB), or five or six bispecific subunits, eachbinding to two different antigens (AB, AB). Thus, the bispecific andmulti-specific IgM pentamers may include five identical bispecificbinding units, monospecific IgM binding units, at least two of them havedifferent binding specificities, or any combination thereof.

A “full length IgM antibody heavy chain” is a polypeptide consisting inN-terminal to C-terminal direction of an antibody heavy chain variabledomain (VH), an antibody constant heavy chain constant domain 1 (CM1 orCμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), anantibody heavy chain constant domain 3 (CM3 or Cμ3), and an antibodyheavy chain constant domain 4 (CM4 or Cμ4). The bispecific full lengthIgM antibodies as defined herein comprise five or six monomers (bindingunits), each with two antigen binding sites, which specifically bind totwo different binding targets (epitopes). The C-terminus of the heavy orlight chain of the full length antibody denotes the last amino acid atthe C-terminus of the heavy or light chain. The N-terminus of the heavyor light chain of the full length antibody denotes the first amino acidat the N-terminus of the heavy or light chain.

Native IgA is a tetrameric protein comprising two identical light chains(κ or λ) and two identical heavy chains (α). In the human, there are twoIgA isotypes, IgA1 and IgA2. IgA, similarly to IgG, contains threeconstant domains (CA1-CA3 or Cα1-Cα3), with a hinge region between theCα1 and Cα2 domains, wherein the “CA” and “Cα” designations are usedinterchangeably. All IgA isotypes have an 18 amino acid “tailpiece”,which is located C-terminal to the Cα3 domain, which enables polymericIg formation (see, e.g. Garcia-Pardo et al., 1981, J. Biol. Chem. 256,11734-11738 and Davis et al., 1988, Eur. J. Immunol. 18, 1001-1008).Serum IgA is a monomer but can also polymerize. In its secretory formIgA comprises from 2-5 of the basic 4-chain units, linked by a J-chain,which may include a tail-piece, and may be associated by a secretorycomponent. The structures of tail-piece, dimeric IgA and secretory IgA,associated with a secretory component (sIgA) are illustrated in FIG. 2.IgA antibodies can be further divided into IgA1 and IgA2 sub-classes.The term “IgA” antibody is used herein to specifically include allsub-classes, i.e. IgA1 and IgA2 antibodies, including dimeric andmultimeric forms, with and without a secretory component, as well asfragments, preferably antigen-binding fragments, of such antibodies. Forthe purposes of the present invention, the IgA antibody preferably is adimer, where two tail-pieces are connected by a J-chain (see, FIG. 2).

The term “IgA” is used herein in the broadest sense and specificallyincludes mono-, and multi-specific IgA molecules, such as, for example,the multi-specific IgA binding molecules disclosed in PCT ApplicationNo. PCT/US2015/015268, the entire disclosure of which is expresslyincorporated by reference herein.

The term “multi-specific IgA” is used herein in the broadest sense torefer to IgA antibodies with two or more binding specificities. Thus,the term “multi-specific” includes “bispecific”, e.g. bispecificantibodies or bispecific binding units, including IgA dimers comprisingtwo monospecific subunits, each binding to a different antigen (AA, BB),or two bispecific subunits, each binding to two different antigens (AB,AB).

In one embodiment, the dimeric multi-specific IgA molecules consist oftwo monospecific binding units, each binding unit having bindingspecificity to a different binding target (AA, BB). In anotherembodiment, in the dimeric IgA molecules at least one of the two bindingunits has two different binding specificities (i.e. is a bispecific,e.g. AA, A,B or AA, BC). In another embodiment, each of the two bindingunits has two specificities, which may be the same (AB, AB) or different(AC, CD or AB, AC, for example).

The term “bispecific IgA antibody binding unit” is used in the broadestsense and specifically covers a pair of IgA antibody heavy chainconstant region polypeptides, comprising at least a CA3 constant domain,fused to a variable domain sequence (V_(H)), each variable domainsequence binding to a different target, with or without associatedantibody light chain variable domain (V_(L)) sequences. In oneembodiment, the bispecific IgA antibody comprises two V_(H)V_(L) antigenbinding regions, each capable of binding to a different epitope on oneantigen or epitopes on two different antigens. The bispecific IgAantibody binding units can be full length from a single species, or bechimerized or humanized.

A “full length IgA antibody heavy chain” is a polypeptide consisting inN-terminal to C-terminal direction of an antibody heavy chain variabledomain (VH), an antibody constant heavy chain constant domain 1 (CA1 orCα1), an antibody constant heavy chain constant domain 2 (CA2 or Cα2),and an antibody heavy chain constant domain 3 (CA3 or Cα3). The bi- ormulti-specific full length IgA antibodies according to the inventioncomprise two monomers (binding units), each of which may be mono- orbispecific, with or without a secretory component. Thus, themulti-specific IgA antibodies of the present invention may includemonospecific and bispecific binding units, provided that the resultantIgA antibody has at least two binding specificities. The C-terminus ofthe heavy or light chain of the full length antibody denotes the lastamino acid at the C-terminus of the heavy or light chain. The N-terminusof the heavy or light chain of the full length antibody denotes thefirst amino acid at the N-terminus of the heavy or light chain.

For further details of the structure and properties of the differentclasses of antibodies, see e.g., Basic and Clinical Immunology, 8thEdition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The term “interface”, as used herein, is used to refer to a region,which comprises those “contact” amino acid residues (or other non-aminoacid groups such as, for example, carbohydrate groups,) in a first IgMheavy chain constant region which interact with one or more “contact”amino acid residues (or other non-amino acid groups) in a second IgMheavy chain constant region.

The term “asymmetric interface” is used to refer to an interface (ashereinabove defined) formed between two antibody chains, such as a firstand a second IgM heavy chain constant region and/or between an IgM heavychain constant region and its matching light chain, wherein the contactresidues in the first and the second chains are different by design,comprising complementary contact residues. The asymmetric interface canbe created by knobs/holes interactions and/or salt bridges coupling(charge swaps) and/or other techniques known in the art, such as forexample, by the CrossMab approach for coupling a u heavy chain to itsmatching light chain. A “cavity” or “hole” refers to at least one aminoacid side chain which is recessed from the interface of the secondpolypeptide and therefore accommodates a corresponding protuberance(“knob”) on the adjacent interface of the first polypeptide. The cavity(hole) may exist in the original interface or may be introducedsynthetically (e.g. by altering nucleic acid encoding the interface).Normally, nucleic acid encoding the interface of the second polypeptideis altered to encode the cavity. To achieve this, the nucleic acidencoding at least one “original” amino acid residue in the interface ofthe second polypeptide is replaced with DNA encoding at least one“import” amino acid residue which has a smaller side chain volume thanthe original amino acid residue. It will be appreciated that there canbe more than one original and corresponding import residue. The upperlimit for the number of original residues which are replaced is thetotal number of residues in the interface of the second polypeptide. Thepreferred import residues for the formation of a cavity are usuallynaturally occurring amino acid residues and are preferably selected fromalanine (A), serine (S), threonine (T), valine (V) and glycine (G). Mostpreferred amino acid residues are serine, alanine or threonine, mostpreferably alanine. In the preferred embodiment, the original residuefor the formation of the protuberance has a large side chain volume,such as tyrosine (Y), arginine (R), phenylalanine (F) or tryptophan (W).

An “original” amino acid residue is one which is replaced by an “import”residue which can have a smaller or larger side chain volume than theoriginal residue. The import amino acid residue can be a naturallyoccurring or non-naturally occurring amino acid residue, but preferablyis the former. By “non-naturally occurring” amino acid residue is meanta residue which is not encoded by the genetic code, but which is able tocovalently bind adjacent amino acid residue(s) in the polypeptide chain.Examples of non-naturally occurring amino acid residues are norleucine,ornithine, norvaline, homoserine and other amino acid residue analoguessuch as those described in Ellman et al., Meth. Enzym. 202:301-336(1991), for example. To generate such non-naturally occurring amino acidresidues, the procedures of Noren et al. Science 244: 182 (1989) andEllman et al., supra can be used. Briefly, this involves chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.The methods of the current invention, in certain embodiments, involvereplacing at least one original amino acid residue in an IgM heavychain, but more than one original residue can be replaced. Normally, nomore than the total residues in the interface of the first or secondpolypeptide will comprise original amino acid residues which arereplaced. The preferred original residues for replacement are “buried”.By “buried” is meant that the residue is essentially inaccessible tosolvent. The preferred import residue is not cysteine to preventpossible oxidation or mispairing of disulfide bonds.

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of thefirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity relies on modeling the protuberance/cavity pair based upon athree-dimensional structure such as that obtained by X-raycrystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art, includingtechniques of molecular modeling.

By “original nucleic acid” is meant the nucleic acid encoding apolypeptide of interest which can be “altered” (i.e. geneticallyengineered or mutated) to encode a protuberance or cavity. The originalor starting nucleic acid may be a naturally occurring nucleic acid ormay comprise a nucleic acid which has been subjected to prior alteration(e.g. a humanized antibody fragment). By “altering” the nucleic acid ismeant that the original nucleic acid is mutated by inserting, deletingor replacing at least one codon encoding an amino acid residue ofinterest. Normally, a codon encoding an original residue is replaced bya codon encoding an import residue. Techniques for genetically modifyinga DNA in this manner have been reviewed in Mutagenesis: a PracticalApproach, M. J. McPherson, Ed., (IRL Press, Oxford, UK. (1991), andinclude site-directed mutagenesis, cassette mutagenesis and polymerasechain reaction (PCR) mutagenesis, for example.

The protuberance or cavity can be “introduced” into the interface of thefirst or second polypeptide by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. According, the protuberance or cavity which is “introduced”is “non-naturally occurring” or “non-native”, which means that it doesnot exist in nature or in the original polypeptide (e.g. a humanizedmonoclonal antibody).

Preferably the import amino acid residue for forming the protuberancehas a relatively small number of “rotamers” (e.g. about 3-6). A“rotamer” is an energetically favorable conformation of an amino acidside chain. The number of rotamers for the various amino acid residuesare reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

Unless stated otherwise, the term “antibody” specifically includesnative human and non-human IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD and IgMantibodies, including naturally occurring variants. Thus, for example,the human IgM sequence is available under GenBank Accession NumberX14940.1, while variants have been reported as GenBank CAB37838.1,CAC20458.1, AFM37312.1, X57331.1, and J00260.1.

The term “native” with reference to a polypeptide (e.g. an antibody or aJ-chain) is used herein to refer to a polypeptide having a sequence thatoccurs in nature, regardless of its mode of preparation. Thus, the terms“native” and “native sequence” are used herein interchangeably, andexpressly encompass recombinant polypeptides with a sequence that isfound in nature.

The term “native sequence J-chain” or “native J-chain” as used hereinrefers to J-chain of native sequence IgM or IgA antibodies of any animalspecies, including mature human J-chain, the amino acid sequence ofwhich is shown in FIG. 3 (SEQ ID NO: 1) and the J-chains of non-humananimal species, including, without limitation, the native sequenceJ-chain polypeptides of SEQ ID NOs: 2 to 44 shown in FIG. 4 or the batJ-chain polypeptide (SEQ ID NO: 45) shown in FIG. 5.

The term “modified J-chain” is used herein to refer to variants ofnative sequence J-chain polypeptides comprising an extraneous bindingmoiety introduced into the native sequence. The introduction can beachieved by any means, including direct or indirect fusion of anextraneous binding moiety or by attachment through a chemical linker.The term “modified human J-chain” specifically encompasses, withoutlimitation, a native sequence human J-chain of the amino acid sequenceof SEQ ID NO: 1 modified by the introduction of a binding moiety. Theterm specifically encompasses, without limitation, a native sequencehuman J-chain of the amino acid sequence of SEQ ID NO: 1 modified by theintroduction of an extraneous binding moiety which does not interferewith efficient polymerization (dimerization) of IgM or IgA and bindingof such polymers (dimers) to a target

The term “binding moiety” is used herein in the broadest sense toencompass any chemical entity capable of specific binding to a target,such as an antigen. Examples of binding moieties include, withoutlimitation, antibodies, antigen-binding fragments of antibodies,antibody-drug conjugates, antibody-like molecules, antigen-bindingfragments of antibody-like molecules, soluble and membrane-boundproteins, ligands, receptors, virus-like particles, protein toxins,enzymes, and alternative scaffolds. Preferred binding moieties arepolypeptides (including peptides), preferably with a biologicalfunction. An exemplary biological function is the ability of a bindingmoiety to bind to and activate an effector cell, such as a B-cell, aT-cell, or a natural killer (NK)-cell.

The term “polypeptide” is used herein in the broadest sense and includespeptide sequences. The term “peptide” generally describes linearmolecular chains of amino acids containing up to about 60, preferably upto about 30 amino acids covalently linked by peptide bonds.

The term “extraneous” with reference to a “binding moiety” is usedherein to refer to a binding moiety not present in a reference nativepolypeptide sequence at the same location. Thus, an extraneouspolypeptide sequence (including peptide sequences), might be comprisedwithin the corresponding native sequence but at a different location. Ina preferred embodiment, the “extraneous” sequence is not present in thecorresponding native sequence in any location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J.Mol. Biol. 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species, while the remainder ofthe chain(s) is identical with or homologous to corresponding sequencesin antibodies derived from another species, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad.Sci. USA 81:6851-6855).

“Humanized” forms of non-human (e.g., murine) antibodies are antibodieswhich contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a hypervariable region ofthe recipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are also replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al. (1986) Nature 321:522-525; Riechmannet al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct.Biol. 2:593-596.

An “isolated” antibody herein is one which has been identified andseparated and/or recovered from a component of its natural environmentin a recombinant host cell. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes, as well asundesired byproducts of the production. In a preferred embodiment, anisolated antibody herein will be purified (1) to greater than 95% byweight, or greater than 98% by weight, or greater than 99% by weight, asdetermined by SDS-PAGE or SEC-HPLC methods, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a amino acid sequencer, or (3) to homogeneity bySDS-PAGE under reducing or non-reducing conditions using Coomassie blueor, preferably, silver stain. Ordinarily, an isolated antibody will beprepare by at least one purification step.

The term “specific binding” or “specifically binds to” or is “specificfor” refers to the binding of a binding moiety to a binding target, suchas the binding of an antibody to a target antigen, e.g., an epitope on aparticular polypeptide, peptide, or other target (e.g. a glycoproteintarget), and means binding that is measurably different from anon-specific interaction (e.g., a non-specific interaction may bebinding to bovine serum albumin or casein). Specific binding can bemeasured, for example, by determining binding of a binding moiety, or anantibody, or an antibody modified by introduction of a binding moiety,to a target molecule compared to binding to a control molecule. Forexample, specific binding can be determined by competition with acontrol molecule that is similar to the target, for example, an excessof non-labeled target. In this case, specific binding is indicated ifthe binding of the labeled target to a probe is competitively inhibitedby excess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about 200nM, alternatively at least about 150 nM, alternatively at least about100 nM, alternatively at least about 60 nM, alternatively at least about50 nM, alternatively at least about 40 nM, alternatively at least about30 nM, alternatively at least about 20 nM, alternatively at least about10 nM, alternatively at least about 8 nM, alternatively at least about 6nM, alternatively at least about 4 nM, alternatively at least about 2nM, alternatively at least about 1 nM, or greater. In certain instances,the term “specific binding” refers to binding where a molecule binds toa particular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.

“Binding affinity” refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). For example, the Kd can be about 200 nM, 150nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM,2 nM, 1 nM, or stronger. Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art.

As used herein, the “Kd” or “Kd value” refers to a dissociation constantmeasured by a technique appropriate for the antibody and target pair,for example using surface plasmon resonance assays, for example, using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at25° C. with immobilized antigen CMS chips at about 10 response units(RU).

The terms “conjugate,” “conjugated,” and “conjugation” refer to any andall forms of covalent or non-covalent linkage, and include, withoutlimitation, direct genetic or chemical fusion, coupling through a linkeror a cross-linking agent, and non-covalent association.

The term “fusion” is used herein to refer to the combination of aminoacid sequences of different origin in one polypeptide chain by in-framecombination of their coding nucleotide sequences. The term “fusion”explicitly encompasses internal fusions, i.e., insertion of sequences ofdifferent origin within a polypeptide chain, in addition to fusion toone of its termini. The term “fusion” is used herein to refer to thecombination of amino acid sequences of different origin

The term “valent” as used herein denotes the presence of a specifiednumber of binding sites in an antibody. As such, the terms “bivalent”,“tetravalent”, and “hexavalent” denote the presence of two bindingsites, four binding sites, and six binding sites, respectively. Thus, ifin a bispecific IgA antibody according to the present invention eachbinding unit is bivalent, the bispecific IgA antibody will have 4valencies.

The term “epitope” includes any molecular determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. A “binding region” isa region on a binding target bound by a binding molecule.

“Polyepitopic specificity” refers to the ability to specifically bind totwo or more different epitopes on the same or different target(s).“Monospecific” refers to the ability to bind only one epitope. Accordingto one embodiment the bispecific IgM antibody binds to each epitope withan affinity of at least 10⁻⁷M, or 10⁻⁸ M or better.

The term “target” or “binding target” is used in the broadest sense andspecifically includes polypeptides, without limitation, nucleic acids,carbohydrates, lipids, cells, and other molecules with or withoutbiological function as they exist in nature.

The term “antigen” refers to an entity or fragment thereof, which canbind to an antibody or trigger a cellular immune response. An immunogenrefers to an antigen, which can elicit an immune response in anorganism, particularly an animal, more particularly a mammal including ahuman. The term antigen includes regions known as antigenic determinantsor epitopes, as defined above.

As used herein, the term “immunogenic” refers to substances, whichelicit the production of antibodies, and/or activate T-cells and/orother reactive immune cells directed against an antigen of theimmunogen.

An “antigen-binding site” or “antigen-binding region” of an antibody ofthe present invention typically contains six complementarity determiningregions (CDRs) which contribute in varying degrees to the affinity ofthe binding site for antigen. There are three heavy chain variabledomain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variabledomain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR and frameworkregions (FRs) is determined by comparison to a compiled database ofamino acid sequences in which those regions have been defined accordingto variability among the sequences and/or structural information fromantibody/antigen complexes. Also included within the scope of theinvention are functional antigen binding sites comprised of fewer CDRs(i.e., where binding specificity is determined by three, four or fiveCDRs). Less than a complete set of 6 CDRs may be sufficient for bindingto some binding targets. Thus, in some instances, the CDRs of a VH or aVL domain alone will be sufficient. Furthermore, certain antibodiesmight have non-CDR-associated binding sites for an antigen. Such bindingsites are specifically included within the present definition.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment Chinese hamsterovary (CHO) cells are used as host cells.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Detailed Description

Design and Production of Antibodies with Modified J-Chain

IgM is the first immunoglobulin produced by B cells in response tostimulation by antigen, and is present at around 1.5 mg/ml in serum witha half-life of 5 days. IgM is a pentameric or hexameric molecule. Justas IgG, IgM monomers consist of two light and two heavy chains. However,while IgG contains three heavy chain constant domains (CH1, CH2 andCH3), the heavy (μ) chain of IgM additionally contains a fourth constantdomain (CH4), similarly to the ε heavy chains in IgE. This extraconstant domain is located in place of the IgG and IgA proline-richhinge region that is responsible for the rotational flexibility of theantigen-binding Fab domains relative to the Fc domain of IgG and IgAantibodies.

Five IgM monomers form a complex with an additional small polypeptidechain (the J-chain) to form a native IgM molecule. The J-chain isconsidered to facilitate polymerization of μ chains before IgM issecreted from antibody-producing cells. While crystallization of IgM hasproved to be notoriously challenging, Czajkowsky and Shao (PNAS106(35):14960-14965, 2009) recently published a homology-basedstructural model of IgM, based on the structure of the IgE Fc domain andthe known disulfide pairings. The authors report that the human IgMpentamer is a mushroom-shaped molecule with a flexural bias. The IgMheavy (μ) chain contains five N-linked glycosylation sites: Asn-171,Asn-332, Asn-395, Asn-402 and Asn-563.

Immunoglobulin A (IgA), as the major class of antibody present in themucosal secretions of most mammals, represents a key first line ofdefense against invasion by inhaled and ingested pathogens. IgA is alsofound at significant concentrations in the serum of many species, whereit functions as a second line of defense mediating elimination ofpathogens that have breached the mucosal surface. Receptors specific forthe Fc region of IgA, FcαR, are key mediators of IgA effector function.Human IgA may have two different IgA heavy constant region (Cα) geneswhich give rise to the two subclasses, IgA1 and IgA2. The maindifference between IgA1 and IgA2 resides in the hinge region that liesbetween the two Fab arms and the Fc region. IgA1 has an extended hingeregion due to the insertion of a duplicated stretch of amino acids,which is absent in IgA2. IgA has the capacity to form dimers, in whichtwo monomer units, each comprising two heavy chains and light chains,are postulated to be arranged in an end-to-end configuration stabilizedby disulfide bridges and incorporation of a J-chain. Dimeric IgA,produced locally at mucosal sites, is transported across the epithelialcell boundary and out into the secretions by interaction with thepolymeric immunoglobulin receptor (pIgR). During this process the pIgRis cleaved and the major fragment, termed secretory component (SC),becomes covalently attached to the IgA dimer.

Both IgA and IgM possess an 18-amino acid extension in the C terminuscalled the “tail-piece” (tp). The IgM (μtp) and IgA (αtp) tail-piecesdiffer at seven amino acid positions. The IgM and IgA tail-piece ishighly conserved among various animal species. The conserved penultimatecysteine residue in the IgA and IgM tail-pieces has been demonstrated tobe involved in polymerization. Both tail-pieces contain an N-linkedcarbohydrate addition site, the presence of which is required for dimerformation in IgA and J-chain incorporation and pentamer formation inIgM. However, the structure and composition of the N-linkedcarbohydrates in the tail-pieces differ, suggesting differences in theaccessibility of the glycans to processing by glycosyltransferases.

The nucleotide and/or protein sequences of J-chains of human, andvarious vertebrate animal species, such as cow, mouse, avian, amphibian,and rabbit, have been reported. The human J-chain contains eightcysteine residues, two (Cys13 and Cys69) are involved in disulfidebridges with the α or μ-chains (in IgA and IgM, respectively), and sixare involved in intrachain disulfide bridges (Cys13: Cys101, Cys72:Cys92, Cys109: Cys134). The three-dimensional crystal structure of theJ-chain has not been reported.

The present invention is based, at least in part, on the recognitionthat the J-chain of IgM and IgA antibodies can be modified byintroduction of a binding specificity (binding moiety), withoutinterfering with the ability of the IgM or IgA antibody to bind to itsbinding target(s). Accordingly, the present invention concerns modifiedJ-chains comprising a binding moiety introduced into a native sequenceJ-chain, such as a native sequence human J-chain of SEQ ID NO: 1. Theinvention further concerns binding molecules comprising a modifiedJ-chain. The binding molecule can, for example, be an IgM antibody, anIgA antibody, or an IgG/IgM or IgG/IgA hybrid antibody, which maycontain an IgM or IgA tail-piece at the IgG heavy chain and thus combinethe properties of IgG and IgA or IgA, including the ability toincorporate and form polymers with a modified J-chain of the presentinvention. For further details on IgG/IgM and IgG/IgA hybrid antibodiessee, e.g. Koteswara et al., Clinical Immunology 2001, 101(1):21-31.

The modified J-chain comprises an extraneous binding moiety, whichincludes, but is not limited to, a polypeptide (including peptides)capable of specifically binding to a binding target, or catalyticcomponents, such as enzyme-like proteases. As discussed earlier, thebinding moieties include, without limitation, antibodies,antigen-binding fragments of antibodies, antibody-drug conjugates,antigen-binding fragments of antibody-drug conjugate, antibody-likemolecules, antigen-binding fragments of antibody-like molecules, solubleand membrane-bound proteins, ligands, receptors, virus-like particles,protein toxins, catalytic components, such as enzymes and enzyme-likeproteases, and alternative scaffolds. It is emphasized that any type ofbinding moiety can be introduced into a J-chain, following the teachingof the present disclosure, by appropriately selecting the location, typeof addition (e.g. direct or indirect fusion, chemical tethering, etc.).

In a preferred embodiment, the binding moiety is an antibody or anantigen-binding fragment of an antibody (also referred to as an“antibody fragment”), including monospecific, bispecific, andmulti-specific antibodies and antibody fragments. The term “antibodyfragment” is used in the broadest sense and includes, withoutlimitation, Fab, Fab′, F(ab′)₂, scFv, and (scFv)₂ fragments,complementarity determining region (CDR) fragments, linear antibodies,single-chain antibody molecules, minibodies, and multispecificantibodies formed from antibody fragments. In a preferred embodiment,the antibody fragment is a scFv.

In another preferred embodiment, the binding moiety is an antibody-likemolecule, such as, for example, a human domain antibody (dAb),Dual-Affinity Re-Targeting (DART) molecule, a diabody, a di-diabody,dual-variable domain antibody, a Stacked Variable Domain antibody, aSmall Modular ImmunoPharmaceutical (SMIP), a Surrobody, astrand-exchange engineered domain (SEED)-body, or T and Ab.

The binding moiety may be a ligand, such as a neurotrophin, aninterleukin, a soluble molecular factor or a growth factor.

Receptors, as binding molecules, include ion-channel-linked,G-protein-linked, and enzyme-linked cell surface receptors. Specificexamples, include, without limitation, ErbB1, ErbB2, ErbB3, ErbB4, TNFR,PDL1, and CTLA-4.

In a further preferred embodiment, the binding moiety is an alternativescaffold. In this context, the term “scaffold” is meant to describe aprotein framework that can carry altered amino acids or sequenceinsertions that confer on protein variants the ability to bind specifictargets. Protein alternative scaffolds include, without limitation,CTLA-4. tendamistat, fibronectin, lipocalins, T-cell receptor, CBM4-2,protein A domain, 1m9, designed AR proteins, designed TPR proteins, zincfinger, pVIII, avian pancreatic polypeptide, GCN4, WW domain, SRChomology domains 2 and 3, PDZ domains, TEM-1, β-lactamase, GFP,thioredoxin, Staphylococcal nuclease, PHD-finger, cl-2, BPTI, APPI,HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, knottins, insectdefensin A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b₅₆₂, LDLreceptor domain A, γ-crystallin, ubiquitin, transferrin, C-typelectin-like domain. Preferred alternative scaffolds are darpins,fibronectin domains and adnectins. For further details see, Binz H K etal, 2005 Nature Biotechnology 23(10):1257-1268.

The binding moiety may be introduced into the native J-chain sequence atany location that allows the binding of the binding moiety to itsbinding target without interfering with the binding of the recipientIgM, IgA, IgG/IgM or IgG/IgA molecule to its binding target or bindingtargets. Preferred locations include at or near the C-terminus, at ornear the N-terminus or at an internal location that, based on thethree-dimensional structure of the J-chain is accessible. In preferredembodiments, the binding moiety is introduced into the native sequenceJ-chain without about 10 residues from the C-terminus or without about10 amino acid residues from the N-terminus, where the native sequenceJ-chain preferably is human J-chain of SEQ ID NO: 1. In anotherembodiment, the binding moiety is introduced into the native sequencehuman J-chain of SEQ ID NO: 1 in between cysteine residues 92 and 101 ofSEQ ID NO: 1, or at an equivalent location of another native sequenceJ-chain. In a further embodiment, the binding moiety is introduced intoa native sequence J-chain, such as a J-chain of SEQ ID NO: 1, at or neara glycosylation site. Most preferably, the binding moiety is introducedinto the native sequence human J-chain of SEQ ID NO: within about 10amino acid residues from the C-terminus.

Introduction can be accomplished by direct or indirect fusion, i.e. bythe combination of the J-chain and binding moiety amino acid sequencesin one polypeptide chain by in-frame combination of their codingnucleotide sequences, with or without a peptide linker. The peptidelinker (indirect fusion), if used, may, for example, be about 1 to 50,or about 1 to 40, or about 1 to 30, or about 1 to 20, or about 1 to 10,or about 10 to 20 amino acid residues, and may be present at one or bothends of the binding moiety to be introduced into the J-chain sequence.In a preferred embodiment, the peptide linker is about 10 to 20, or 10to 15 amino acids long. In another preferred embodiment, the peptidelinker is 15 amino acids long.

The binding moiety may also be appended to the native J-chain sequenceby chemical linkage using heterobifunctional protein crosslinkerscontain two different functional groups, which have their own reactivityand selectivity. These crosslinkers can be used in a one step process orcan be used to create activated proteins, which can often be preservedand reacted with the second biomolecule in a separate step. Thus, forexample, a heterobifunctional crosslinking reagent can be used to formconjugates between a J-chain and a binding moiety. The reactive groupsinclude, without limitation imine reactive groups (such as NHS orSulfo-NHS), maleimide groups, and the like. Such crosslinkers, which canbe cleavable or non-cleavable, have been used, for example, in theformation of hapten carrier proteins and in preparing enzyme-antibodyconjugates. Chemically, the cleavable crosslinkers specifically include,without limitation, disulfide-based, hydrazone, and peptide linkers. Awell known and much studied enzyme-labile linker is a valine-citrullinelinker but other peptide linkers are also known and suitable. Typicalrepresentatives of non-cleavable linkers include thioethers, such asSMCC (N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate).For further details see, e.g. Ducry L and Stump B, Bioconjugate Chem.2010, 21:5-13, the entire disclosure of which is expressly incorporatedby reference herein. For listing of further suitable linkers see, e.g.Klein et al., Protein Engineering, Design & Selection; 2014,27(10):325-330, the entire disclosure of which is expressly incorporatedby reference herein.

While the modified J-chain usually contains one extraneous bindingmoiety, it is also possible to introduce more than one binding moietyinto a J-chain.

The modified J-chain may be produced by well known techniques ofrecombinant DNA technology, by expressing nucleic acid encoding themodified J-chain in a suitable prokaryotic or eukaryotic host organism,such as CHO cells or E. coli. Thus, the modified J-chain may, forexample, be expressed in E. coli, as described by Symersky et al., MolImmunol 2000, 37:133-140.

In one embodiment, the J-chain can be initially modified by insertion ofan enzyme recognition site, and post-translationally modified by apeptide or non-peptide linker, which can tether any extraneous bindingmoiety to the J-chain, such as, for example, cytotoxic small molecule tomake an antibody-drug conjugate (ADC).

The modified J-chain can also be co-expressed with the heavy and lightchains of the recipient IgM, IgA, IgG/IgM or IgG/IgA antibody. Althoughdue to its complex structure, the large scale production of recombinantIgM has been difficult, several recombinant production systems for IgMusing non-lymphoid cells have been reported, including co-expression ofthe IgM heavy (H) and light (L) chains in C6 glioma cells, CHO cells,and HeLa cells (see, e.g. W089/01975 and Wood et al., J. Immunol. 145,3011-3016 (1990) for expression in CHO cells). Expression of an IgMmonoclonal antibody in E. coli, with or without a J-chain, is described,e.g. in Azuma et al., Clin Cancer Res 2007, 13(9):2745-2750. Productionof IgM in an immortalized human retina cell line expressing E1A and E1Bproteins of an adenovirus is described in U. S. Application PublicationNo. 20060063234.

The recipient antibody may be monospecific, bispecific ormulti-specific. Bispecific and multi-specific IgM and IgA bindingmolecules, including antibodies, are described, for example, in U.S.Application Ser. Nos. 61/874,277 and 61/937,984, the entire contents ofwhich are hereby expressly incorporated by reference.

Applications of Antibodies with Modified J-Chain

Antibodies comprising a modified J-chain of the present invention havewidespread therapeutic and diagnostic applications.

In one embodiment, antibodies comprising a modified J-chain bind to twoor more sites on the same soluble target, such as, for example, VEGF,TNFα, or IL6. The purpose may, for example, be antagonizing multiplesites on the protein and/or increasing the avidity to a given target.

In another embodiment, the antibodies comprising a modified J-chainherein bind two or more sites on the same cell surface (receptor)target, such as EGFR or HER2 (ErbB2). Thus, for example, such antibodiesmight target both the 4D5 and the 2C4 epitopes on a HER2 molecule. Thisapproach may increase bio-potency and/or avidity to a given target.

In yet another embodiment, the antibodies comprising the modifiedJ-chains of the present invention bind two or more different solubletargets (globular proteins or peptides), e.g. TNFα and IL6, VEGFα andAng2, or two cytokines. This approach might result, for example, in morecomplete blocking a specific pathway; blocking of the so called“cytokine storm,” i.e. undesirable T cell activation resulting fromcertain multivalent bispecific antibodies, such as bispecific antibodiesfor the CD3 antigen, or coordinate an enzyme and its substrate, e.g.Factor IXa and Factor X. Specific examples include, without limitation,bispecific antibodies with modified J-chain, where a first specificityis directed to VEGF and a second specificity is directed to Ang2 or DLL4(anti-angiogenesis), or a first specificity is directed to TNF and asecond specificity is directed to Ang2 or IL-17 (anti-inflammatoryproperties), where either specificity may be introduced into the J-chainof an IgM, IgA, IgG/IgM or IgG/IgA antibody, or an antigen-bindingfragment thereof.

In a further embodiment, antibodies comprising a modified J-chain maybind one or more soluble targets and one or more cell surface receptortargets, such as an angiogenic factor and neo-vascular specificreceptor. The purpose of this approach may also be increased deliveryand blockade at specific sites or tissues.

In a still further embodiment, antibodies comprising a modified J-chainare designed to bind two or more different cell surface receptortargets, such as, for example, HER1, HER2 (ErbB2) and HER3 (ErbB3),inhibiting multiple targets through the same or different pathways. Thismay result in enhancing specificity and selectivity and/or in morecomplete blocking of a given pathway. Specific examples of suchantibodies include, without limitation, bispecific antibodies with amodified J-chain where one specificity is directed to HER2 and anotherspecificity is directed to HER3; or one specificity is directed to EGFR(HER1) and another specificity is directed to HER2. Other bispecificIgM, IgA, IgG/IgM or IgG/IgA antibodies with a modified J-chain may, forexample, bind to EGFR and HER3, IL-1α and IL-1β, IL-4 and IL-13, Ang-2and VEGF-A, Factor IXA and Factor X, or IL-17A and IL-17F.

Antibodies comprising a modified J-chain of the present invention mayalso be designed to bind one or more soluble targets or cell surfacereceptor targets and one or more long residence time targets, such as,for example, TNFα and/or VEGF and serum albumin. These molecules areexpected to have longer circulating half-life than binding moleculeswithout the albumin specificity.

In a further embodiment, antibodies comprising a modified J-chain hereinmay bind one or more soluble targets and one or more matrix proteinsand/or substrates, such as, for example, VEGFα and hyaluronic acid. Theresultant multi-specific binding molecules may find utility, forexample, in anti-angiogenic therapy of ocular conditions, such asage-related macular degeneration (AMD), due to their increased residencetime in the intraocular space.

Antibodies comprising a modified J-chain and binding one or more solubleor receptor target, plus one or more transporter receptor (ietransferrin receptor), e.g. anti-EGFRvIII (mutant form with exon IIIdeleted) found glioblastoma combined with anti-transferrin specificity,can find utility in antibody delivery across blood brain barrier.

In a preferred embodiment, the IgM, IgA, IgG/IgM and IgG/IgA antibodiesherein comprise a modified J-chain with binding specificity for animmune cell, such as a T-cell, NK-cell, a macrophage, or a neutrophil,and bind to an antigen expressed on a disease cell or pathogen. Since anIgM molecule comprises 5 binding units, and an IgA molecule is a dimercomprising two binding units, such molecules are significantly morepotent due to their greater avidity than bispecific IgG antibodies. Inaddition, by activating and redirecting effector cells, e.g. effector Tcells, to targeted disease cells, tissues or pathogens the IgM, IgA,IgG/IgM and IgG/IgA antibodies herein induce an immune response againstthe target, thereby further increasing potency and efficacy. Due tothese beneficial properties, the IgM, IgA, IgG/IgM and IgG/IgAantibodies herein, comprising a modified J-chain, are especiallyadvantageous in situations where IgG antibodies bind to their targetwith low affinity. Thus, in one embodiment, the IgM, IgA, IgG/IgM andIgG/IgA antibodies herein may comprise the binding domain of atherapeutic IgG antibody.

In certain embodiments, the IgM, IgA, IgG/IgM and IgG/IgA antibodiesherein comprising a modified J-chain may be used for the treatment ofcancer. It is anticipated that any type of tumor and any type oftumor-associated antigen may be targeted. Exemplary types of cancersinclude, without limitation, acute lymphoblastic leukemia, acutemyelogenous leukemia, biliary cancer, breast cancer, cervical cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectalcancer, endometrial cancer, esophageal, gastric, head and neck cancer,Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin'slymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer. However, the skilled artisan will realize thattumor-associated antigens are known in the art for virtually any type ofcancer.

Tumor-associated antigens that may be targeted by the IgM, IgA, IgG/IgM,or IgG/IgA antibodies of the presence invention include, withoutlimitation, alpha-fetoprotein (AFP), associated antigens, Ba 733, BAGE,BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m,CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132,CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4,CXCR7, CXCL12, HIF-1a, colon-specific antigen-p (CSAp), CEA (CEACAM5),CEACAM6, c-Met, DAM, EGFR (HER1, ErbB1), ErbB2 (HER2), ErbB4 (HER3),EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growthfactor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100,GRO-.beta., HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and itssubunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M,HST-2, Ia, IGF-1R, IFN-γ IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP,MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreaticcancer mucin, PD-1 receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6,IL-25, RSS, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-.alpha., Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras.

As discussed above, for oncological applications, antibodies comprisinga modified J-chain can be designed to trigger the killing function ofimmune cells against targeted cancer cells. Thus, for example, theJ-chain of IgM, IgA, IgG/IgM or IgG/IgA antibodies, or antigen-bindingfragments thereof, can be modified by introducing into a native J-chaina binding specificity for an immune cell, such as a T-cell or a NaturalKiller (NK) cell, while the IgA, IgG/IgM or IgG/IgA antibody providesbinding specificity to a target cell, e.g. a tumor marker, such as anoncogene, on the surface of a tumor cell, including, for example, any ormore of the tumor-associated antigens listed above.

In the case of T-cells, cluster of differentiation 3 (CD3) is amultimeric protein complex, known historically as the T3 complex, and iscomposed of four distinct polypeptide chains (ε, γ, δ, ζ) that assembleand function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complexserves as a T cell co-receptor that associates non-covalently with the Tcell receptor (TCR). Components of this CD3 complex, especially CD3ε,are targets for a modified J-chain of an IgM antibody specificallybinding to a tumor-associated antigen. Although the modified J-chainspecific for effector T cells preferably binds to the CD3 (CD3ε)antigen, other antigens expressed on effector T cells are known and maybe targeted by the modified J-chain. Exemplary T-cell antigens include,but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30,CD40, CD40L, CD44, CD45, CD69 and CD90.

Exemplary IgM, IgA, IgG/IgM, or IgG/IgA antibodies including a modifiedJ-chain with CD3 binding specificity, may include the binding regions ofknown IgG antibodies to tumor-associated antigens, such as, for example,blinatumomab (also known as MT103) (anti-CD19), CD19hA19 (anti-CD19,U.S. Pat. No. 7,109,304), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567),hA20 (anti-CD20, U.S. Pat. No. 7,251,164), hIMMU31 (anti-AFP, U.S. Pat.No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2(anti-CD22, U.S. Pat. No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No.7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14(anti-CEACAM5, U.S. Pat. No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat.No. 7,541,440), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3(anti-CEACAM6, U.S. Pat. No. 7,541,440), Ab124 and Ab125 (anti-CXCR4,U.S. Pat. No. 7,138,496), the disclosures of which are expresslyincorporated by reference herein.

In a specific embodiment, an IgM, IgA, IgG/IgM, or IgG/IgA antibodycomprising the CD19 binding region of blinatumomab comprises a modifiedJ-chain comprising the CD3 binding region of blinatumomab. This antibodycan be used, e.g. for the treatment of non-Hodgkin lymphoma, or acutelymphoblast leukemia of the B cell series (B-ALL).

In another specific embodiment, an IgM, IgA, IgG/IgM, or IgG/IgAantibody comprising the CD20 binding region of rituximab comprises amodified J-chain with CD3 specificity, such as a modified J-chaincomprising the CD3 binding region of blinatumomab.

In yet another specific embodiment, an IgM, IgA, IgG/IgM, or IgG/IgAantibody comprising the EpCAM binding region of MT110 comprises amodified J-chain with CD3 specificity, such as a modified J-chaincomprising the CD3 binding region of MT110. Such bispecific antibodiescan be used for the treatment of gastrointestinal cancer.

Alternative antibodies that can provide binding regions for use incombination with a modified J-chain with CD3 binding specificityinclude, for example, abciximab (anti-glycoprotein IIb/IIIa),alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR),gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab(anti-EGFR), tositumomab (anti-CD20), trastuzumab (anti-ErbB2),lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor),ipilimumab (anti-CTLA-4), abagovomab (anti-CA-125), adecatumumab(anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125),obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026(anti-PSMA, U.S. patent application Ser. No. 11/983,372, deposited asATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575),tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab(anti-CD25), efalizumab (anti-CD11 a), GA101 (anti-CD20; Glycart Roche),atalizumab (anti-.alpha.4 integrin), omalizumab (anti-IgE);anti-TNF-.alpha. antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), BENLYSTA® (HumanGenome Sciences); antibodies for therapy of Alzheimer's disease such asAlz 50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47),gantenerumab, solanezumab and infliximab; anti-fibrin antibodies like59D8, T2G1s, MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG),MOR202 (Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson &Johnson); anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971),Ab 75, Ab 76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90),as well as the anti-HIV antibodies described in U.S. Pat. No. 5,831,034,U.S. Pat. No. 5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170and Joos et al., Antimicrob. Agents Chemother. 2006; 50(5):1773-9.

FIG. 19 lists exemplary target antigens for the treatment of hematologiccancer, epithelial tumor, and glycosyl-rich tumors, and exemplaryantigens on T-cells, NK-cells, macrophages and neutrophils for targetingwith a modified J-chain. It is to be understood that an IgM moleculebinding to any of the listed tumor antigens can be combined with amodified J-chain with any of the listed binding specificities. Thus, anyantibody target listed in the left column of the table can be combinedwith any modified J-chain target listed in the right column.

In a preferred embodiment, an IgM pentamer provides binding specificityto target cells, such as B-cells, while a binding domain to an effectorcell, e.g. a T cell can be covalently attached to the J-chain of the IgMantibody. Thus, the J-chain can be modified by covalent attachment of aCD3 (CD3ε) binding domain. In this configuration, the IgM pentamer(comprising 10 copies of heavy chain and 10 copies of light chain)provides binding specificity to the target B-cells, while the T-celltethering of the J-chain delivers cytotoxic potency. In other words, theIgM antibody binding to a tumor target additionally acquires a T-cellbinding function. The CD3 binding domain covalently attached to a nativeJ-chain, or a variant of a native J-chain, can, for example be asingle-chain Fv (scFv) of an anti-CD3 antibody, or a naturally occurringheavy chain only antibody, e.g. a camelid (camels, llamas, alpacas) orsingle-chain antibody of cartilaginous fish (sharks, rays), a scaffold,e.g. fibronectin (e.g. fibronectin III) with CD3 binding specificity.While certain preferred embodiments are specifically referred to herein,it is to be understood that IgM, IgA, IgG/IgM and IgG/IgA antibodieswith binding specificity to any target, such as any tumor antigen,comprising a modified J-chain binding to any T-cell marker arecontemplated and are within the scope of the present invention.

In one embodiment, a multi-specific IgM, IgA, IgG/IgM or IgG/IgAantibody binds to one or more of the tumor targets listed above, whilethe J-chain is modified to bind to CD3ε. In a preferred embodiment, themulti-specific IgM, IgA, IgG/IgM or IgG/IgA antibody binds to one ormore of the tumor targets listed in FIG. 19, while the J-chain ismodified to bind to CD3ε.

Natural killer (NK) cells are important components of the innateimmunity and play a key role in host defense by virtue of their abilityto release cytokines and to mediate cytolytic activity against tumorcells and virus-infected cells. NK cell antigens include, withoutlimitation, CD16, CD32a, CD56, CD57, CD64, CD117 (or c-kit), adhesionmolecules including lymphocyte-associated molecule-2 (LFA-2 or CD2),LFA-3 (CD58), and LFA-1 (CD11a/CD18).

Examples of NK cell engaging bispecific antibodies with modified J-chaininclude IgM, IgA, IgG/IgM and IgG/IgA antibodies with bindingspecificity to any of the tumor antigens listed above comprising amodified J-chain binding to an NK cell. In a particular embodiment, abispecific IgM, IgA, IgG/IgM and IgG/IgA antibody with HER2 bindingspecificity comprises a J-chain modified to bind CD16, CD32a, CD56, orCD64. In another preferred embodiment, a multi-specific IgM, IgA,IgG/IgM or IgG/IgA antibody binds to any of the tumor targets listed inthe left column of the table in FIG. 19, and the J-chain is modified tobind CD16, CD64 or NKG2D on NK cells.

Macrophage engagement in the J-chain modified antibodies of the presentinvention can be provided, for example, by introducing a CD14specificity into the J-chain.

In one embodiment, a multi-specific IgM, IgA, IgG/IgM or IgG/IgAantibody binds to one or more of the tumor targets listed above, whilethe J-chain is modified to bind to CD14. In a preferred embodiment, themulti-specific IgM, IgA, IgG/IgM or IgG/IgA antibody binds to one ormore of the tumor targets listed in FIG. 19, while the J-chain ismodified to bind to CD14.

The IgM, IgA, IgG/IgM and IgG/IgA antibodies with modified J-chain cantarget carbohydrate-based antigens, see, e.g. a review article by Cazetet al., Breast Cancer Research; 2010, 12:204. Carbohydrate-based tumorantigens have been shown to have good tumor association, withalternative forma of glycosylation that allow the production of IgGantibodies binding to such antigens with reasonable specificity but notnecessarily high affinity. Using IgM, IgA, IgG/IgM, or IgG/IgAantibodies with associated increased avidity against this class ofantigens represents great opportunities for new therapeutic antibodies,especially coupled with effector cell mobilization achieved by J-chainmodification. In one preferred embodiment, the IgM, IgA, IgG/IgM andIgG/IgA antibody binds to one or more carbohydrate based tumor antigen,while the J-chain is modified to bind any of the effector cells listedin FIG. 19.

In another preferred embodiment, the IgM, IgA, IgG/IgM and IgG/IgAantibodies with modified J-chain can be used as part of IgM, IgA,IgG/IgM or IgG/IgA antibodies directed against pathogens. In a preferredembodiment, the pathogens are selected from the group consisting of HIVvirus, Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpessimplex virus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reovirus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, West Nile virus, Plasmodiumfalciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei,Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Elmeriatenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata,Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M.hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivariumand M. pneumoniae, as disclosed in U.S. Pat. No. 6,440,416.

In this embodiment, the immune effector cell may, for example, be aneutrophil.

FIG. 19 specifically lists viral antigens as targets for the IgM, IgA,IgG/IgM and IgG/IgA antibodies herein, in combination with neutrophilmarkers which can be targeted by the modified J-chain of suchantibodies.

It is noted that the IgM, IgA, IgG/IgM and IgG/IgA antibodies herein mayalso target carbohydrate antigens on carbohydrate rich cancers. Suchantigens include CEA, CA-125, TADG78, Sialyl Lewis-X (CD15), forexample.

In all embodiments, the binding moiety (binding unit) used to modify anative J-chain may be introduced before or after the J-chain. Thus, amodified J-chain with CD3 binding specificity may have ananti-CD3_(scFv)-J or a J-anti-CD3_(scFv) configuration. The sequence(SEQ ID NO: 46) and structure of an anti-CD3 single chain Fv comprisingthe J-chain at the C-terminus (anti-CD3_(scFv)-J) is shown in FIG. 8.The sequence (SEQ ID NO: 47) and structure of an anti-CD3 single chainFv comprising the J-chain at the N-terminus (J-anti-CD3_(scFv)) is shownin FIG. 9. Due to their increased avidity such antibodies are superiorrelative to bispecific IgG antibodies. For example, as a result, theyare suitable for targeting low copy-number targets, such as Rituxanresistant Burkitt lymphoma cells characterized by low level of CD20expression. In addition, the IgM, IgA, IgG/IgM and IgG/IgA antibodiesherein comprising a modified J-chain have greatly enhanced potencyrelative to bispecific IgG antibodies.

Pharmaceutical Compositions of Antibodies with Modified J-Chain

For therapeutic uses, antibodies comprising a modified J-chain may beformulated into pharmaceutical compositions. A pharmaceuticalcomposition of the present invention can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thetarget disease or condition and the desired results. To administer acompound of the invention by certain routes of administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation. For example, the compound may beadministered to a subject in an appropriate carrier, for example,liposomes, or a diluent. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Pharmaceutical carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art.

The compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and/or dispersing agents. Preventionof presence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

The following examples sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Further details of the invention are illustrated by the following,non-limiting Examples.

EXAMPLE 1 Preparation of a Bispecific Anti-CDIM IgM Antibody Comprisinga Modified J-chain Binding CD3

1. Generation of DNA Constructs with Designed Mutations

-   -   a. DNA construct synthesis. All the DNA constructs with designed        mutations are synthesized by the commercial vendors        (Genescript), with compatible restriction sites at both ends for        subcloning into respective expression vectors.    -   b. Constructing expression vectors. The synthesized DNA        constructs are re-suspended in Tris-EDTA buffer at 1 μg/ml. DNA        (1 μg) is subjected to enzyme digestion and the synthesized gene        is separated from the carrier plasmid DNA by electrophoresis.        The digested DNA is ligated to pre-digested plasmid DNA (pCAGGS        for J-chain, Gene 108 (1991) 193-200) by standard molecular        biology techniques. The ligated DNA is transformed into        competent bacteria and plated on LB plates with multiple        selective antibiotics. Several bacterial colonies are picked and        DNA preparations are made by standard molecular biology        techniques. The prepared DNA are verified by sequencing. Only        the bacterial clones with 100% match of DNA sequence with the        designed DNA sequence are used for plasmid DNA preparation and        subsequently for cell transfection.    -   c. Different J-chains. In order to demonstrate that J-chain        variants will be able to couple with IgM, two different J-chain        variants are constructed with distinct fusion sites        incorporating anti-CD3 antibody (OKT3 scFv).        -   i. This construct is composed of a scFv of OKT3 (anti-CD3)            fused with N-terminus of human J-chain (CD3scFv-15 aa            Linker-J):

(SEQ ID NO: 46) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGSEQKL ISEEDLNSAVDHHHHHH--

This construct has a molecular weight about 45kD and able to bind tosoluble epsilon chain of CD3 (Sino Biological), or T cells; and is ableto bind to anti-myc monoclonal antibody 4A6 or other anti-mycantibodies.

-   -   ii. This construct is composed of a scFv of OKT3 (anti-CD3)        fused with C-terminus of human J-chain (J-15 aa Linker-CD3scFv):

(SEQ ID NO: 47) QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIKEQKLISEE DLNSAVDHHHHHH-

This J-CD3scFv construct has a molecular weight about 45kD and is ableto bind to soluble epsilon chain of CD3 (Sino Biological), or T cells;and is able to bind to anti-myc monoclonal antibody 4A6 or otheranti-myc antibodies.

-   -   d. IgM heavy chain: This heavy chain construct has a full length        μ chain for IGM-55.5 which binds CDIM on the surface of B-cells:

(SEQ ID NO: 3) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRMAWGASVNFDYWGQGTLVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

This heavy chain construct has a molecular weight about 64 kD and whenco-expressed with light chain, the resultant IgM is able to bind to CDIMpositive B cells.

-   -   e. Light chain for IGM-55.5 known as IGM-55.5, which binds CDIM        (cell death inducing molecule) on the surface of B-cells:

(SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The light chain construct has a molecular weight about 24 kD and whenco-expressed with the appropriate heavy chain (SEQ ID NO 3) is able tobind to CDIM positive B cells.

2. Protein Expression, Purification and Characterization

-   -   a. Transfection. Heavy, Light and Modified J-chain DNA is        transfected into CHO cells. DNA for expression vectors are mixed        typically in 1:1:1 ratio with PEI and then added to CHO-S cells.        PEI transfection with CHO-S cells is conducted according to        established techniques (see “Biotechnology and Bioengineering,        Vol 87, 553-545”).    -   b. Immunoprecipitation        -   i. Capture Select IgM (BAC, Thermo Fisher). IgM proteins            from transfected CHO cell supernatants are partially            purified by immuno-precipitation with Capture Select IgM            affinity matrix according to manufacturers' protocol (GE            Life Sciences). After incubation at room temperature for 2            hours, the affinity matrix is separated from the supernatant            by centrifugation. The matrix is further washed with PBS for            3 times before the PBS is carefully removed. The captured            protein is eluted from the matrix by incubating with NuPage            LDS protein buffer (Life Technology) for 5 minutes.        -   ii. Anti-myc agarose affinity matrix (Sigma). IgM proteins            from transfected CHO cell supernatants are partially            purified by immunoprecipitation with anti-myc affinity            matrix according to manufacturers' protocol. After            incubation at room temperature for 2 hours, the affinity            matrix is separated from the supernatant by centrifugation.            The matrix is further washed with PBS for 3 times before the            PBS is carefully removed after the final wash. The captured            protein is eluted from the matrix by incubating with NuPage            LDS protein buffer (Life Technology) for 5 minutes.    -   c. Gel electrophoresis        -   i. Non-reducing SDS PAGE        -   ii. Non-reducing SDS PAGE separates native IgM and its            mutant forms according to size. Pentameric IgM, composed of            homodimeric heavy and light chains, produces a protein band            of approximately 1,000,000 molecular weight. NuPage LDS            Sample Buffer (Life Technologies) is added to IgM protein            samples at 25 C for 30 minutes before loading onto the gel.            NativePage Novex 3-12% Bis-Tris Gel (Life Technologies) is            used with Novex Tris-Acetate SDS Running Buffer (Life            Technologies). Run gel until the dye front reaches the            bottom of the gel.        -   iii. Reducing SDS-PAGE. NuPage LDS sample buffer (Life            Technologies) and NuPage reducing agent dithiothreitol (Life            Technologies) are added to IgM protein samples and heated to            80° C. for 10 minutes before loading on NuPage Novex 4-12%            Bis-Tris Gel (Life Technologies). NuPage MES SDS Running            Buffer (Life Technologies) is used for gel electrophoresis.            Gels are run until the dye front reaches the bottom of the            gel. After electrophoresis is complete, remove gel from            apparatus and stain the gel using Colloidal Blue Staining            (Life Technologies).        -   iv. Western Blot Detection. After electrophoresis is            complete, remove gel from XCell SureLock Mini-Cell. Transfer            to PVDF membrane at 30 volts for 1 hour (refer to Life            Technologies' manual). Block with 20 ml 3% BSA in PBST at 25            C for 1 hour.        -   For anti-J-chain Western blot, add anti-J (SP105, Thermo            Fisher) at 1:500 in 3% BSA in PBST overnight at 4C. Wash            with PBST four times at room temperature. Add HRP-Goat anti            rabbit IgG (Jackson Immunology) at 1:5,000 in 3% BSA in PBST            for 1 hour at room temperature. Wash with PBST 4 times at            room temperature. Add 10 ml of HRP chemiluminescent            substrate (Thermo Fisher) for 10 minutes before exposing the            blot to film. Anti-J-chain antibody only reacts with IgM            which is co-expressed with either unmodified J-chain            (FIG. 6) or modified J-chain (FIG. 7).        -   For anti-myc western blot, add anti-myc (4A6, Millipore) at            1:200 in 3% BSA in PBST for overnight at 4C. Wash with PBST            four times at room temperature. Add HRP-Goat anti-mouse IgG            (Jackson Immunology) at 1:5,000 in 3% BSA in PBST for 1 hour            at room temperature. Wash with PBST 4 times at room            temperature. Add 10 ml of HRP chemiluminescent substrate            (Thermo Fisher) for 10 minutes before exposing the blot to            film. Anti-myc antibody only reacts with modified J-chain            with myc tag (FIG. 2).

3. Bi-Specific Functional Analysis

-   -   a. FACS analysis of target binding        -   IGM-55.5 with CD3scFv-J or IGM-55.5 with J-CD3scFv binding            to T cells is confirmed by binding of antibody to T cell            line (Jurkat, CD3 positive cell line) and B cell line            (Nalm6, negative control cell line). After washing,            rhodamine labeled 4A6 is added to the cell suspension. The            cell target binding is detected by MFI of both positive and            negative control cells with or without CD3 antigen. IGM-55.5            with CD3scFv-J or IGM-55.5 with J-CD3scFv binding to CDIM            expressing cells is confirmed by binding of antibody to            positive cell line (Daudi, positive cell line) and negative            cell line (Peer). After washing, rhodamine labeled 4A6 is            added to the cell suspension. The cell target binding is            detected by MFI of both positive and negative control cells            with or without CDIM antigen.    -   b. FACS analysis of T-cell mediated B-cell killing in co-culture        -   Raji, a CD19+CD20+ B cell line, was co-cultured with T-ALL,            a CD8+ Cytolytic T cell line with CD20 IgM×J-wild-type or            CD20 IgM×CD3-J chain for 24 hours at 37 degrees, 5% CO2.            Cell were harvested and stained with fluorescent antibodies            to CD3 (552852/BD Biosciences) and CD19 (555413/BD            Biosciences) and analyzed by flow cytometry to assess viable            B cells.    -   c. Complement dependent cytotoxicity of B-cells        -   Ramos, a CD20+ cell line was seeded in 96 well half area            white plates at 25,000 cells/well. Antibody and human            complement (5% final, Quidel) were added to initiate            complement dependent cytotoxicity and the number of viable            cells were measured using Cell Titer Glo and manufacturer's            protocol. Luminescence was measured on an Envision multimode            reader (Perkin Elmer) using 0.1 s integration time per well.            Percent viable cells was calculated by normalizing the            luminescence values (Relative luminescence units—RLU) versus            wells with no added drug. Data were analyzed using GraphPad            Prism and a four parameter fit with top and bottom values            fixed at 100 and 0% viability respectively.

Multi-specific binding and multi-specific functional analysis can beperformed in a similar manner using techniques known in the art, such asthose described above.

EXAMPLE 2

Molecular Cloning, Expression and Purification of a Anti-CD20 IgMAntibody with a Modified J-chain Carrying an Anti-CD3 Binding scFv

This example describes the preparation of the molecular cloning,expression and purification of a further IgM antibody targeting adifferent B-cell antigen (CD20) and a modified J-chain binding to CD3.The DNA corresponding to the heavy and light chain sequences below wasprepared using the methods as described in Example 1.

SEQ ID NO: 48: IgM Light chain sequence of the anti-CD20 antibody(rituximab) MDMRVPAQLLGLLLLWLRGARCQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 49:IgM Heavy chain sequence of the anti-CD20 antibody (rituximab)MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

The DNA corresponding to these heavy and light chains as well as thatcorresponding to either the wild-type (wt) J-chain, O15J or J15O J-chainsequences described in Example 1 were co-transfected into HEK293 cellsand proteins expressed and purified using the camelid resin as describedbefore. As shown in FIG. 13, all four proteins express well. Theanti-CD20 IgM hexamer without J-chain is clearly resolved from theJ-chain containing pentamers for the IgM pentamer with the wild typeJ-chain as well as for the bispecific IgM's where the anti-CD3 scFv islinked to the J-chain in either orientation (O15J or J15O).

EXAMPLE 3

Molecular Cloning, Expression and Purification of Bispecific Anti-CD20IgM Antibody Comprising a Modified J-chain Carrying a Different Anti-CD3Binding scFv

To establish that assembly of bispecific IgM is feasible with a modifiedJ-chain carrying an anti-CD3 scFv of a different sequence than that usedExamples 1 and 2, a J-chain carrying the variable regions from theantibody Visilizumab (Nuvion) was performed. Shown below are thesequences for two J-chains with the scFv corresponding to Visilizumab(V) fused to the J-chain through a linker containing 15 aa's in twodifferent orientations—V15J and J15V.

SEQ ID NO: 50: J chain sequence for V15JMGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMV ETALTPDACYPDSEQ ID NO: 51: J-chain sequence for J15VMKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWS SNPPTFGGGTKLEIK

As described in Example 1, DNA corresponding to these sequences wassynthesized and transfected into HEK293 cells along with the heavy andlight chains for anti-CD20 IgM to produce protein which was thenpurified using the camelid antibody affinity matrix specific for IgM. Asshown in FIG. 13, J-chains fused to the new anti-CD3 scFv with the 1.5aa linker are able to incorporate into the IgM and the pentameric formof bi-specific IgM with the corresponding J-chain is clearlydistinguishable from the hexameric form without a J-chain.

EXAMPLE 4 Molecular Cloning, Expression and Purification of an IgMComprising an Anti-CD16 Camelid Modified J-chain Versions of a SecondEffector Cell Binding Domain—Anti-CD16 Camelid

This example demonstrates that it is possible to link a binding moietydifferent from an scFv to a J-chain, which is directed to a differenttarget on a different effector cell population. A camelid Vhh sequenceshown below was used, that was selected for binding to the CD16 antigenon natural killer cells (NK cells). Once again, this sequence was linkedto a J-chain using a flexible 15 aa linker to produce C15J. Thebispecific IgM was expressed and purified as described in Example 1 andanalyzed on hybrid gels. Formation of a pentameric species is clearlyseen. Further, incorporation of the C15J J-chain into the pentameric IgMwas established using western blot (FIG. 18).

SEQ ID NO: 52: J-chain sequence for C15JMGWSYIILFLVATATGVHSEVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

EXAMPLE 5

Analysis of Complement Dependent Cytotoxicity for Family of IgM's withand without Incorporated J-chains

Complement dependent cytotoxicity is a key mechanism for cell killing byantibodies. IgM antibodies are known to have enhanced complementdependent cell killing (CDC) due to their multimeric form. A key aspectof this invention was to test if incorporation of modified J-chains,which carry scFv or camelid. Vhh binders of effector cells at eithertheir C- or N-termini, causes interference with binding of C1q—the keycomponent of the complement pathway, and therefore may inhibit CDC. Wemeasured the CDC activity of each of the IgM and bispecific IgMconstructs. As shown in FIG. 14, we find that incorporation of themodified J-chain has, unexpectedly, no deleterious effect on the CDCactivity of our bispecific IgM's, Moreover, with the linker lengths wetested, we find that the bispecific IgM's have CDC activity between60-100 fold enhanced over the corresponding IgG on a molar basis (FIG.14).

EXAMPLE 6 Analysis of T-cell Dependent B-cell Killing by Bispecific IgMin In Vitro Co-Culture

Engagement of effector T-cells by bispecific IgM antibodies with amodified J-chain is expected to greatly enhance killing of the targetB-cell populations compared to the IgM carrying no J-chain or the wildtype J-chain. To test cell killing in co-culture, we performed a cellkilling assay as described in Example 1. Antibody at a single highconcentration (100 ng/mL) incubated with CD20+ Raji cells and CD3+effector T-ALL cells. As shown in FIG. 15, the bispecific IgM carrying aCD3 binding scFv on its J-chain is able to cause complete killing ofB-cells. The same experiment done as a dose response shows that there isa greater than 300 fold improvement in B-cell killing compared to theIgM that carries no scFv capable of engaging T-cells. Complete killingof B-cells by bispecific IgM is observed at concentrations as low as 10ng/mL.

EXAMPLE 7 Demonstration of T-cell Dependent B-cell Killing In Vivo inNSG Mouse Model

In order to test the bispecific IgM's we made in an in vivo context, weperformed experiments with humanized non-obese diabetic severe combinedimmune-deficient gamma null (NSG) mice. These mice have severelyimpaired immune function and lack mouse T- and B-lymphocytes. They arereconstituted with human CD34+ stem cells to create mice withessentially human lymphocyte populations. When CD20 IgM×CD3-J chain wasdosed intravenously in these animals at 0.5 mg/kg and whole blood wasobtained and analyzed by flow cytometry for circulating levels of humanB cells, we observed a complete depletion of the B-cell population evenwith treatments as short as 6 hours (FIG. 17).

While several embodiments have been provided in the present disclosure,it should be understood that the disclosed systems and methods might beembodied in many other specific forms without departing from the spiritor scope of the present disclosure. The present examples are to beconsidered as illustrative and not restrictive, and the intention is notto be limited to the details given herein. Various examples of changes,substitutions, and alterations are ascertainable by one skilled in theart and could be made without departing from the spirit and scopedisclosed herein.

1. An IgM antibody comprising a modified J-chain, wherein the modifiedJ-chain comprises one or more extraneous binding moieties introducedinto a native sequence J-chain.
 2. The IgM antibody of claim 1, whereinthe native sequence J-chain is the native human J-chain sequence of SEQID NO: 1, or a functional fragment thereof.
 3. The IgM antibody of claim2, wherein the native sequence J-chain is the native human J-chainsequence of SEQ ID NO:
 1. 4. The IgM antibody of claim 3, wherein theextraneous binding moiety is introduced into the native human J-chainsequence of SEQ ID NO: 1 by direct or indirect fusion.
 5. The IgMantibody of claim 4, wherein the binding moiety is introduced byindirect fusion through a peptide linker.
 6. The IgM antibody of claim5, wherein the indirect fusion is through a peptide linker at aC-terminus of the extraneous binding moiety.
 7. The IgM antibody ofclaim 5, wherein the indirect fusion is through a peptide linker at anN-terminus of the extraneous binding moiety.
 8. The IgM antibody ofclaim 6, wherein the extraneous binding moiety is introduced into thenative human J-chain sequence of SEQ ID NO: 1 at a C-terminus of thenative human J-chain sequence.
 9. The IgM antibody of claim 7, whereinthe extraneous binding moiety is introduced into the native humanJ-chain sequence of SEQ ID NO: 1 at an N-terminus of the native humanJ-chain sequence.
 10. The IgM antibody of claim 5, wherein theextraneous binding moiety is introduced into the native human J-chainsequence in between cysteine residues 92 and 101 of SEQ ID NO:
 1. 11.The IgM antibody of claim 5, wherein said peptide linker has a lengththat ranges from 1 to 20 amino acids.
 12. The IgM antibody of claim 11,wherein the length of the peptide linker is 15 amino acids.
 13. The IgMantibody of claim 3, wherein the extraneous binding moiety is introducedinto the native human J-chain sequence of SEQ ID NO: 1 by chemical orchemo-enzymatic derivatization.
 14. The IgM antibody of claim 13,wherein the extraneous binding moiety is introduced into the nativehuman J-chain sequence of SEQ ID NO: 1 by a chemical linker.
 15. The IgMantibody of claim 14, wherein the chemical linker is a cleavable ornon-cleavable linker.
 16. The IgM antibody of claim 15, wherein thecleavable linker is a chemically labile linker or an enzyme-labilelinker.
 17. The IgM antibody of claim 15, wherein the linker is selectedfrom the group consisting of: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), iminothiolane (IT), bifunctional derivatives ofimidoesters, active esters, aldehydes, bis-azido compounds,bis-diazonium derivatives, diisocyanates, bis-active fluorine compounds,and any combination thereof.
 18. The IgM antibody of claim 13, modifiedby insertion of an enzyme recognition site, and by post-translationallyattaching the extraneous binding moiety at the enzyme recognition sitethrough a peptide or non-peptide linker.
 19. The IgM antibody of claim1, wherein the one or more extraneous binding moieties are selected fromthe group consisting of: antibodies, antigen-binding fragments ofantibodies, antibody-drug conjugates, antibody-like molecules,antigen-binding fragments of antibody-like molecules, soluble andmembrane-bound proteins, ligands, receptors, virus-like particles,protein toxins, enzymes, and alternative scaffolds.
 20. The IgM antibodyof claim 19, wherein the alternative scaffold is selected from the groupconsisting of: darpins, fibronectin domains, adnectins, and knottins.21. The IgM antibody of claim 19, wherein the antigen-binding fragmentis selected from the group consisting of: F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, scFv, and single domain antibody.
 22. The IgM antibody of claim 21,wherein the antigen-binding fragment is an scFv.
 23. The IgM antibody ofclaim 1, which is monospecific.
 24. The IgM antibody of claim 1, whichis bispecific.
 25. The IgM antibody of claim 1, which is multi-specific.26. A composition comprising an IgM antibody comprising a modifiedJ-chain, wherein the modified J-chain comprises one or more extraneousbinding moieties introduced into a native sequence J-chain.
 27. Thecomposition of claim 26, which is a pharmaceutical composition, furthercomprising a pharmaceutically acceptable carrier.
 28. The composition ofclaim 26, which is a diagnostic composition.
 29. A method of treating acancer or viral disease, the method comprising administering to asubject in need of treatment an effective amount of an IgM antibodycomprising a modified J-chain, wherein the modified J-chain comprisesone or more extraneous binding moieties introduced into a nativesequence J-chain.